Size-dependent isolation of primordial germ cells from avian species.

Primordial germ cells (PGCs), the precursors of sperm or ova, could be used to generate transgenic animals and interspecies germ-line chimeras, which would facilitate the recovery of endangered species by making their access and manipulation in vitro easier. During early embryogenesis in avian species, PGCs are transported via the bloodstream to the gonadal anlagen. PGCs of most avian species-particularly wild or endangered birds-are not readily isolated from the embryonic bloodstream because germ-cell markers have not yet been defined for them. Here, we report a rapid, efficient, and convenient method for PGC isolation from various avian species. Blood PGCs were isolated based on the difference in size between PGCs and other blood cells, using a microporous membrane. The efficiency of this size-dependent isolation for the White Leghorn chicken was not significantly different from that of magnetic-activated cell sorting, and the isolated cells expressed chicken PGC-related genes and PGC-specific markers. The utility of the method was then verified in Japanese quail (Coturnix japonica), Mallard duck (Anas platyrhynchos), and Muscovy duck (Cairina moschata). Immunocytochemistry and an in vivo migration assay indicated that this method was able to enrich for true embryonic blood PGCs without specific antibodies, and could be applied to the development of avian interspecies chimeras for restoration of wild or endangered species.

Inactivation of the tumor suppressor WTX in a subset of pediatric tumors.

WTX is a tumor suppressor gene expressed during embryonic development and inactivated in 20-30% of cases of Wilms tumor, the most common pediatric kidney cancer. WTX has been implicated in several cellular processes including Wnt signaling, WT1 transcription, NRF2 degradation, and p53 function. Given that WTX is widely expressed during embryonic development and has been recently shown to regulate mesenchymal precursor cells in several organs, we tested for the potential involvement of WTX in a panel of pediatric tumors and adult sarcomas. A total of 353 tumors were screened for WTX deletions by fluorescence in situ hybridization (FISH). Discrete somatic WTX deletions were identified in two cases, one hepatoblastoma and one embryonal rhabdomyosarcoma, and confirmed by array comparative genomic hybridization. Direct sequencing of the full WTX open reading frame in 24 hepatoblastomas and 21 embryonal rhabdomyosarcomas did not identify additional mutations in these tumor types. The presence of WTX mRNA was confirmed in hepatoblastomas and embryonal rhabdomyosarcomas without WTX deletions by RNA-in situ hybridization. Notably, tumors with evidence of WTX inactivation, Wilms tumor, hepatoblastoma and rhabdomyosarcoma, are primitive tumors that resemble undifferentiated precursor cells and are linked to overgrowth syndromes. These results indicate that WTX inactivation occurs in a wider variety of tumor types than previously appreciated and point to shared pathogenic mechanisms between a subset of pediatric malignancies.

Nuclear clusterin is associated with neuronal apoptosis in the developing rat brain upon ethanol exposure.

BACKGROUND: Fetal alcohol spectrum disorder (FASD) is often accompanied by reduced brain volumes, reflecting brain cell death induced by ethanol, but the molecular mechanisms were less elucidated. This study was set up to investigate whether clusterin (Clu) was involved in neuronal cell death in developing rats. METHODS: Seven-day-old rats were subcutaneously injected with 20% ethanol in normal saline at 3 g/kg twice. The upregulation of Clu and cell death was detected by immunohistochemistry, immunofluorescence microscopy, and/or Western blotting. Protein-protein interaction was detected by immunoprecipitation and immunoblotting. To identify the isoform interacting with Bcl-XL, HT22 mouse hippocampal cells were transfected with nuclear Clu(nClu)- or secretory Clu-expressing vector, and confocal microscopy was performed. Clu transcripts were knocked down in primary cortical cells using siRNA. RESULTS: We found that Clu increased in the cerebral cortex following acute ethanol treatment. The Clu upregulation was related to increased cell death, which was assessed by activated caspase-3 and TUNEL staining. The upregulated Clu was a nuclear isoform that was nuclear translocated upon ethanol exposure and interacted with Bcl-XL, mediating apoptosis. CONCLUSIONS: This study shows that nClu plays a pro-apoptotic role in ethanol-induced cell death in the developing brain, providing new insights for development of FASD.

Dosimetry of a novel converging X-ray source for kilovoltage radiotherapy.

PURPOSE: The objective of this work was to evaluate phantom dosimetry of a novel kilovoltage (kV) X-ray source, which employs a stationary tungsten anode and a linearly swept scanning electron beam. The source utilizes converging X-ray collimation along with orthogonal mechanical rotation to distribute surface flux over large area. In this study, this was investigated as a potential solution to fast-falloff limitations expected with kV radiotherapy. This was done with the aim of future clinical development of a lower cost radiotherapy alternative to megavoltage (MV) linac systems. METHODS: Radiochromic film was employed for dosimetry on the kV X-ray source of the linear-converging radiotherapy system (LCRS). The source utilizes charge particle optics to magnetically deflect and focus an electron beam along a stationary, reflection tungsten target in an ultra-high-vacuum stainless-steel chamber. Resulting X-rays were collimated into converging beamlets that span a large planar angle and converge at the system isocenter. In this study, radiochromic film dosimetry was done at 140 and 145 kVp for a designated planning treatment volume (PTV) of 4 cm diameter. An acrylic phantom was employed for dose distribution measurements of stationary and rotational delivery. Film dosimetry was evaluated in planes parallel to the source X-ray window at various depths, as well as in the plane of gantry rotation. RESULTS: At 140 and 145 kVp and using a collimated 4 cm square field at depth, lesion-to-skin dose ratio was shown to improve with additional beams from different relative source positions, where the different beams are focused at the same isocenter and do not overlap at the phantom surface. It was only possible to achieve a 1:1 Dmax -to-surface ratio with four delivery beams, but the ratio improved to 4:1 with 12 beams, focused at the same isocenter depth of 7.8 cm in an acrylic phantom. For the tests conducted, the following Dmax -to-surface ratios were obtained: 0.4:1 lesion-to-skin ratio for stationary delivery from one entry beam, 0.71:1 lesion-to-skin ratio was obtained for two beams, 1.07:1 ratio for four beams, and 4:1 for 12 beams. Dose-depth profiles were evaluated for stationary and rotational dosimetry. Additionally, rotational dosimetry was measured for a case more analogous to a clinical scenario, where the isocenter was located at an off-center simulated lesion. CONCLUSIONS: The results demonstrate potential dose-depth improvements with kV arc therapy by distributing the surface flux with a wide converging beam along with perpendicular mechanical source rotation of the LCRS. The system delivered tolerable dose to a large surface area when a threshold of multiple, separated beams was reached. The radiochromic film data support the feasibility of the construct of the LCRS kV radiotherapy system design.

Evaluation of combinatorial cis-regulatory elements for stable gene expression in chicken cells.

BACKGROUND: Recent successes in biotechnological application of birds are based on their unique physiological traits such as unlimited manipulability onto developing embryos and simple protein constituents of the eggs. However it is not likely that target protein is produced as kinetically expected because various factors affect target gene expression. Although there have been various attempts to minimize the silencing of transgenes, a generalized study that uses multiple cis-acting elements in chicken has not been made. The aim of the present study was to analyze whether various cis-acting elements can help to sustain transgene expression in chicken fibroblasts. RESULTS: We investigated the optimal transcriptional regulatory elements for enhancing stable transgene expression in chicken cells. We generated eight constructs that encode enhanced green fluorescent protein (eGFP) driven by either CMV or CAG promoters (including the control), containing three types of key regulatory elements: a chicken lysozyme matrix attachment region (cMAR), 5'-DNase I-hypersensitive sites 4 (cHS4), and the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE). Then we transformed immortalized chicken embryonic fibroblasts with these constructs by electroporation, and after cells were expanded under G418 selection, analyzed mRNA levels and mean fluorescence intensity (MFI) by quantitative real-time PCR and flow cytometry, respectively. We found that the copy number of each construct significantly decreased as the size of the construct increased (R2 = 0.701). A significant model effect was found in the expression level among various constructs in both mRNA and protein (P < 0.0001). Transcription with the CAG promoter was 1.6-fold higher than the CMV promoter (P = 0.027) and the level of eGFP expression activity in cMAR- or cHS4-flanked constructs increased by two- to three-fold compared to the control CMV or CAG promoter constructs. In addition, flow cytometry analysis showed that constructs having cis-acting elements decreased the level of gene silencing as well as the coefficient of variance of eGFP-expressing cells (P < 0.0001). CONCLUSIONS: Our current data show that an optimal combination of cis-acting elements and promoters/enhancers for sustaining gene expression in chicken cells is suggested. These results provide important information for avian transgenesis and gene function studies in poultry.

Selective decrease of chick embryonic primordial germ cells in vivo and in vitro by soft X-ray irradiation.

The feasibility of soft (low-energy) X-ray irradiation as a means of depleting the endogenous primordial germ cell(s) (PGC) of chicken embryos, to improve the efficiency of germ cell-mediated transgenesis, was investigated. Eggs were subjected to a non-irradiated control treatment and embryos were exposed for 40s to soft X-ray at 15, 16.5, or 18 kV ( approximately 1.5, 1.65, and 1.8 Gy, respectively). Exposure of stage X embryos to each dose of X-ray resulted in a reduction of approximately 50% in the number of PGC apparent at stage 28, whereas the total number of gonadal cells was unaffected. Irradiation (16.5 kV) of embryos at stage 9 or 14 also resulted in similar decreases in the number of PGC with no effect on the total number of gonadal cells. Irradiation did not affect embryo hatchability, compared with the non-irradiated control treatment, although the hatch rate increased with the age of embryos at the time of irradiation. Exposure of gonadal cells isolated from stage 28 embryos to X-ray (16.5 kV, approximately 0.8 Gy) prevented the increase in PGC number during subsequent culture for 10 days; the increase in the total number of gonadal cells was not affected. In conclusion, exposure of chicken embryos to a low dose of soft X-rays is effective for depleting the endogenous PGC population without affecting embryo hatchability or somatic cell viability.

Expressed sequence tag analysis of Eimeria-stimulated intestinal intraepithelial lymphocytes in chickens.

Intraepithelial lymphocytes (IELs) play a critical role in protective immune response to intestinal pathogens such as Eimeria, the etiologic agent of avian coccidiosis. A list of genes expressed by intestinal IELs of Eimeria-infected chickens was compiled using the expressed sequence tag (EST) strategy. The 14,409 ESTs consisted of 1851 clusters and 7595 singletons, which revealed 9446 unique genes in the data set. Comparison of the sequence data with chicken DNA sequences in GenBank identified 125 novel clones. This EST library will provide a valuable resource for profiling global gene expression in normal and pathogen-infected chickens and identifying additional unique immune-related genes.

Development of a hamster superovulation program and adverse effects of gonadotropins on microfilament formation during oocyte development.

OBJECTIVE: To establish a superovulation procedure for the golden hamster (Mesocricetus auratus) by elucidating gonadotropin effects on oocyte development. DESIGN: Randomized, prospective study. SETTING: University laboratory of embryology and gamete biotechnology. ANIMAL(S): Twelve- to 15-week-old female and sexually mature male hamsters. INTERVENTION(S): Different doses of pregnant mare serum gonadotropin (PMSG) were injected into female hamsters in metestrus, diestrus, or proestrus. The same dose of hCG was injected 56 hours later. MAIN OUTCOME MEASURE(S): Embryo development and oocyte morphology after treatment. RESULT(S): First, 10 IU or 15 IU each of PMSG and hCG was injected into 10 hamsters weighing <110 or 110-130 g, respectively. All hamsters were mated, but none delivered live young after injection. Second, the doses of 15 IU, 7.5 IU, 5 IU, or 0 IU of each gonadotropin were injected into each hamster (regardless of body weight, 5 per each group). Increasing numbers of embryos were retrieved as the dosage was increased (11.2 to 46.6 embryos per hamster), whereas the percentage of two-cell embryos at retrieval was significantly decreased (100% to 3%, P<.05). In subsequent culture, none developed to blastocysts after 15-IU injection, whereas 47%, 55%, and 70% of two-cell embryos developed after 7.5-IU, 5-IU, and 0-IU treatments, respectively. As a result, females injected with 5 IU yielded more blastocysts than did females without injection (67 vs. 39). The number of inner cell mass cells per blastocyst was greatly increased in the control groups compared with the 5-IU and 7.5-IU treatment groups (22 vs. 14.3-14.7 cells per blastocyst). Third, the ultrastructure of oocytes was examined after injecting 5 IU each of PMSG and hCG (regardless of body weight). Superovulation did not affect oocyte maturation, but different patterns in microfilament formation were detected after the treatment. CONCLUSION(S): Female hamsters can be superovulated effectively by injecting equal amounts of PMSG and hCG, 56 hours apart. However, embryo development was adversely affected in a dose-dependent manner at all doses of gonadotropins, and microfilament distribution was affected by such treatment.

An interclass nuclear transfer between fowl and mammal: in vitro development of chicken-to-cattle interclass embryos and the detection of chicken genetic complements.

An attempt was made to develop an interclass somatic cell nuclear transfer method as an alternative means of establishing chicken embryonic stem cells. Chicken-to-cattle interclass embryos that activated calcium ionophore, cycloheximide, and cytochalasin D were developed into blastocysts, and the developing interclass embryos had chicken genetic complements.

Development of a nonmechanical enucleation method using x-ray irradiation in somatic cell nuclear transfer.

Irradiation of in-vitro-matured bovine oocytes with x-rays of different durations was performed to develop an alternative to conventional mechanical enucleation methods in somatic cell nuclear transfer. No significant difference in embryo development to the blastocyst stage was detected between nonmechanical and mechanical methods, and cytologic analyses of karyotype and microtubule formation showed the potential availability of x-ray irradiation.

Electrical conductivity imaging by magnetic resonance electrical impedance tomography (MREIT).

Magnetic resonance electrical impedance tomography (MREIT) is a recently developed imaging technique that combines MRI and electrical impedance tomography (EIT). In MREIT, cross-sectional electrical conductivity images are reconstructed from the internal magnetic field density data produced inside an electrically conducting subject when an electrical current is injected into the subject. In this work the results of an electrical conductivity imaging experiment are presented, along with some practical considerations regarding MREIT. The MREIT experiment was performed with a 0.3 Tesla MRI system on a phantom made of two compartments with different electrical conductivities. The current density inside the phantom was measured by the MR current density imaging (MRCDI) technique. The measured current density was then used for conductivity image reconstruction by the J-substitution algorithm. The conductivity phantom images obtained with an injection current of 28mA showed conductivity errors of about 25.5%.